1. Field of the Invention
The present invention generally relates to a process and apparatus for obtaining viable sterile primary cells from tissue, and more particularly relates to a semiautomated process and apparatus useful for obtaining live, sterile, tissue cells from chick or duck embryos or the like. In addition, the present invention relates to an improved centrifuge means useful in separating enzymatically isolated cells from a liquid in which they are carried, and further relates to an improved shredding device for mechanically breaking down the tissue prior to digestion with an enzyme. The present invention also relates to a process for shredding the tissue.
2. Description of the Prior Art
The obtaining of cells from tissue, as for example chick or duck embryos, is known per se and produces materials which are useful in biochemical or immunological research. Typically, such tissue cells are obtained by breaking eggs which contain embryos and extracting the partially developed embryos therefrom. The embryos are then cut up into small pieces and washed in a washing solution. Following the washing step, the minced embryos are mixed for a period of time at a controlled temperature with an enzyme such as trypsin, which breaks down the intercellular material and releases the individual cells or groups thereof. Following this enzyme digestion step, the cells are separated from the enzyme solution by any suitable means such as by spinning the mixture in a bucket type, fixed volume, centrifuge which separates the solid from the liquid material. The cellular material recovered from the centrifuge may then be maintained alive until it is used for culture purposes or the like.
The prior art teaches a manual technique for recovering cells from tissue such as chick or duck embryos. The process, for example, involves hand cutting or mincing the whole embryo, such as with scissors, followed by washing the same with the washing liquid, thereafter mixing the minced pieces of embryo with the enzyme (such as trypsin) in a flask, with the contents being stirred therein with the use of, for example, a magnetic stirrer device. After a suitable amount of time in which the minced embryos are digested with the enzyme at a controlled temperature, the resulting mixture is poured into a glass flask and subjected to centrifugation to separate the solid cellular material from the liquid, after which the liquid is decanted from the glass flask. The remaining solid cellular material may then be mixed with a nutrient to maintain the cellular material viable for a suitable period of time prior to use.
The prior art has recognized that the production of large amounts of tissue cells from chick embryos by manual techniques is difficult and expensive. Thus, F. C. Belton and B. P. Garriock (in "A Semi-Automatic Method for the Production of Primary Cell Suspensions," Journal of Applied Chemistry and Biotechnology, Volume 22, pages 335-341 [1972]) have suggested a tissue production complex useful for processing chick embryo suspensions. The apparatus described in that article consists of a ram-type cutter for mincing the washed whole embryos, a processing unit in which enzyme digestion may be conducted, and a plurality of bucket type centrifuges for separating the cellular material from a solution thereof. However, the system disclosed by Belton and Garriock lacks the degree of automation of the present invention. Further, for reasons to be explained hereinafter, that prior system is incapable of obtaining the high cell yields which the present invention obtains.
Obviously, any open system for recovering the tissue cells is difficult and time consuming and the possibility of contamination of the cells is substantial. For a general review of various techniques used in recovering live tissue cells, reference is here made to Cells and Tissues in Culture, edited by E. N. Willmer, Academic Press, Inc., New York (1965).
Despite the teachings of the prior art, there is a need for a process and apparatus useful for obtaining the tissue cells which is capable of handling large numbers of embryos while producing good yields with minimal handling of the same during processing and thereby decreasing the possibility of contaminating the cells during the operations.